Lysosomes are one of the most important cellular organelles in breast cancer invasion, metastasis, and extracellular matrix (ECM) remodeling, because they contain several degradative enzymes that can influence the integrity and structure of the ECM. The roles of lysosomes in the breast cancer degradome in vivo are virtually unexplored, due, in part, to a lack of noninvasive imaging capabilities available to visualize lysosomes. We recently developed and validated novel near-infrared fluorescent (NIRF) probes for noninvasive imaging of lysosomes in cell culture and tumors in vivo. Preliminary data obtained using these probes support the importance of lysosomes in breast cancer invasion and metastasis. We therefore intend to use these probes to understand and define the influence of lysosomes in cancer cells on invasion, metastasis, and ECM remodeling. Collagen type I (Col1) fibers are an important component of the ECM that can facilitate breast tumor formation, invasion, and metastasis. In this application, we will perform multi-scale molecular and functional imaging studies to characterize the Col1 fiber matrix in primary tumors and metastatic nodules to understand how Col1 fibers are shaped by the cancer degradome, and how this promotes invasion and metastasis. We will microscopically image Col1 fibers using innovative and clinically translatable optical second harmonic generation (SHG) microscopy, which detects an intrinsic optical signal from Col1 fibers. We will utilize the lysosome-specific NIRF probe IR-2 to image lysosomes in breast tumor models in vivo. We will optically image protease activities in vivo using MMPSense to detect matrix metalloprotease activities and ProSense to detect cathepsin activities. We will focus on understanding the role of lysosomes in breast tumor invasion in vivo in Aim 1, and in breast cancer metastasis in Aim 2 using multi-scale optical imaging approaches. In Aim 3, we will determine the influence of lysosomes and lysosomal enzymes on Col1 fiber structure and integrity in solid tumors and metastatic nodules in vivo. These three Aims will provide novel insights into the role of lysosomes and lysosomal enzymes within the overall degradome in breast cancer models. The proposed research will further our understanding of the role of lysosomes in cancer invasion, metastasis, and Col1 fiber remodeling, which may eventually lead to novel lysosome-based therapeutic strategies to treat cancer. Understanding the role of lysosomes and lysosomal enzymes in invasion, metastasis, and Col1 fiber remodeling may lead to the development of innovative diagnostic imaging techniques that noninvasively detect lysosomal probes as surrogate markers to assess invasiveness, metastatic potential, and Col1 fiber structure in these tumors.